Abstract
Caspase-2 is a unique and conservative cysteine protease which plays an important role in several cellular processes including apoptotic cell death. Although the molecular mechanisms of its activation remain largely unclear, a major role belongs to the architecture of the caspase-2 active center. We demonstrate that the substitution of the putative phosphorylation site of caspase-2, Serine-384 to Alanine, blocks caspase-2 processing and decreases its enzymatic activity. Strikingly, in silico analysis using molecular dynamics simulations has shown that Serine-384 is crucially involved in interactions within the caspase-2 active center. It stabilizes Arginine-378, which forms a crucial hydrogen bond with the aspartate residue of a substrate. Hence, Serine-384 is essential for supporting a proper architecture of the active center of caspase-2. Moreover, molecular modeling strongly proved steric inaccessibility of Ser-384 to be phosphorylated. Importantly, a multiple alignment has demonstrated that both Serine-384 and Arg-378 residues are highly conservative across all members of caspase family, which allows us to suggest that this diade is indispensable for caspase processing and activity. Spontaneous mutations in this diade might influence oncosuppressive function of caspases, in particular of caspase-2. Likewise, the mutation of Ser-384 is associated with the development of lung squamous cell carcinoma and adenocarcinoma. Taken together, we have uncovered a central feature of the caspase-2 activation mechanism which is crucial for the regulation of its signaling network.
Highlights
Apoptosis is an evolutionarily conserved program that regulates the homeostasis and development of multicellular organisms
We studied the effect of Ser-384Ala substitution on apoptotic cell death by Annexin V/Propidium iodide staining (AnV/propidium iodide (PI)) in combination with flow
A multiple alignment of the primary structure of initiator, executor and inflammatory caspases and comparison of their tertiary structure demonstrated a high conservation of both Ser-384 and Arg-378 residues across the caspase family (Fig. 6h) and across different species (Fig. 6g). This analysis supports an important role of the Ser-384/Arg-378 interaction for the proper molecular architecture of the active center of caspase-2. These results clearly showed that the Ser-384 residue plays a crucial role in substrate binding and is necessary for caspase-2 processing and enzymatic activity to induce apoptotic cell death
Summary
Apoptosis is an evolutionarily conserved program that regulates the homeostasis and development of multicellular organisms. Upon DNA damage, cells could undergo apoptosis via the death receptor and/or the intrinsic mitochondrial pathway[3]. Caspase-2 is an evolutionarily conserved member of the family and possesses several unique characteristics, including the presence of a nuclear localization signal, which plays a central role in the triggering of apoptotic pathways after DNA damage in some cellular models. Caspase-2 has been reported to be indispensable for the proper cell proliferation, genomic stability, response to ER and oxidative stress[8]. This protein negatively regulates necroptosis and performs a number of non-apoptotic functions[9,10]. Caspase-2, as one of the initiator caspases, has a long prodomain which
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